MAP Kinases are effector kinases that orchestrate diverse but coordinated responses to extracellular signals. In addition they play an essential role in malignant transformation in response to virtually all oncogenes. Upon activation, MAP Kinases move to sites of action where they phosphorylate proteins, thus altering their activity and bringing about the Changes in growth, death, differentiation and function appropriate for the particular cell-agonist interaction. Although the importance of MAP Kinases in cellular physiology is well-established, few functionally significant substrates have been unambiguously identified. To address this problem, we propose three major Specific Aims: I. Engineering Mutant MAP Kinase. We propose to engineer MAP kinase mutants capable of utilizing orthogonal ATP analogs as tools to identify the primary substrates of MAP kinase. II. CENP-E ani Other Mitotic Substrates of MAP Kinase. We will begin by analyzing the primary substrates for MAP Kinase in mitotic Chromosomes, since we have found that a portion of the active MAP kinase in mitosis is in kinetochores, and we have identified the microtubule motor protein CENP-E as a candidate substrate. We also will analyze the mechanism by which MAP Kinases stably associate with these structures, mapping the interaction domains on kinase and substrate. The functional importance of these phosphorylations will be assessed where appropriate. III. Activation pathway for MAP Kinase in G2 and M. We will ask what the signaling pathway is that activates MAP Kinase in mitosis. Active MAP Kinase in other times and places. In addition to the three major Aims, we propose preliminary studies to extend this investigational paradigm to other sites of localization of active MAP Kinase, in particular the nucleus during interphase.